A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the 'Kinnow mandarin', a commercial citrus crop cultivated in the northwest of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcription polymerase chain reaction (RT-PCR) that is time consuming. Here. we describe a novel, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. To standardize the RT-LAMP assay, four different primers were designed and tested to target the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green L The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that arc common in India. The ICRSV RT-LAMP assay developed in the present study is a simple, rapid, sensitive, specific technique. Moreover, the assay consists of only a single step and is more cost effective than existing methods. This is the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large-scale indexing of field samples in diagnostic laboratories, in nurseries, and for quarantine applications.